线粒体肌病非编码RNA表达谱研究及潜在分子标志物筛选

发布时间：2018-02-23 20:38

本文关键词： 线粒体病线粒体脑肌病非编码RNA A3243G miRNA lncRNA 标志物　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：线粒体DNA(mtDNA)或/和编码线粒体功能相关蛋白的核DNA发生基因缺失或点突变,造成编码线粒体氧化代谢必需的酶或载体发生障碍,糖原和脂肪酸等不能进入线粒体充分利用,导致能量代谢障碍和产生复杂的临床症状称为线粒体疾病(mitochondrial disease)。线粒体疾病骨骼肌受累者称为线粒体肌病(mitochondrial myopathy)。线粒体肌病涉及临床类型复杂,包括线粒体肌病伴高乳酸血症和卒中样发作(MELAS)综合征等多种临床综合征,不同临床类型往往具有不同致病基因突变但亦有重叠。围绕线粒体与疾病及衰老、细胞程序性死亡、氧自由基生成以及细胞信号转导调控过程中的相互关系的研究一直是线粒体医学的研究热点。基因测序的进步让我们意识到,人类基因组存在着数以万计的多种虽不具有编码蛋白的功能但具有基因调控和表观遗传修饰功能的非编码RNA(ncRNA),如微小非编码 RNA(miRNA)、长链非编码 RNA(long non-coding RNA,lncRNA)。近年来ncRNAs己被证明可以通过多种不同机制影响基因的表达水平,但绝大多数非编码RNA的功能还是未知的。更为复杂的是各类RNA分子相互间存在复杂的调控网络。得益于核酸测序等技术的飞速发展,越来越多研究发现在人类血清/血浆中存在大量稳定的ncRNA。非编码RNA分子具备可以作为疾病生物学标志物的各类特征,有巨大潜力成为线粒体肌病等疾病的诊治标志物。线粒体功能相关ncRNA的研究还只是处于起步阶段,ncRNA如何影响线粒体基因表达及其功能是一个全新的领域。越来越多的研究证实非编码RNA可以通过影响核基因编码线粒体相关蛋白基因表达或直接在线粒体中调节线粒体自身基因表达影响线粒体的功能。目前线粒体功能相关非编码RNA研究仍大多局限在体外线粒体损伤细胞模型相关非编码RNA的发现及功能验证。尽管已发现多种影响线粒体功能的miRNAs和lncRNAs,但ncRNA调控线粒体功能详细机制有待进一步阐明。ncRNA与线粒体肌病的相关研究尚未见报道,我们有必要探究ncRNA参与线粒体肌病发生发展的的详细机制,或能更深入地揭示ncRNA与线粒体功能的密切联系,且为线粒体肌病找到潜在的治疗靶点。我们希望能在ncRNA调控层面,在线粒体肌病中发现调控线粒体功能或者参与线粒体肌病致病机制的某些关键ncRNAs,进而可以通过影响ncRNA实现异常线粒体的有效清除等影响线粒体肌病发生发展的关键病理过程的调控。具备疾病诊治标志物特征的ncRNA分子具备还可以作为线粒体肌病诊治标志物服务临床。为此,本课题通过筛选线粒体肌病患者肌肉组织和血清中与健康对照者差异表达的ncRNA(miRNA/lncRNA),构建不同RNA之间相互调控网络,探索ncRNA参与线粒体肌病发生发展的潜在通路及线粒体损伤对关键性IncRNA的表达调控机制,并寻找线粒体肌病发生发展过程中的关键ncRNA(miRNA/lncRNA),为ncRNA用于线粒体肌肉病的诊断和治疗提供前期理论依据。本课题围绕着线粒体病非编码RNA参与线粒体肌病发生发展的调控机制进行了以下几方面的工作:第一部分线粒体肌病肌肉非编码RNA(mi RNA/IncRNA)差异表达谱研究研究目的采用高通量技术结合后续RT-PCR重复性验证的前期筛选策略,筛选代表性线粒体脑肌病MELAS(A3243G突变患者)肌肉组织中非编码RNA(miRNA/lncRNA)的差异表达谱,为后续寻找感兴趣的ncRNA以及探究非编码RNA参与线粒体肌病的复杂机制提供前期基础。研究对象和方法1)首先选取MELAS(A3243G突变患者)及活检排除肌肉病变的对照者各20例,采用miRNA表达谱芯片和IncRNA高通量测序技术,筛选MELAS(A3243G突变患者)相比对照肌肉组织中非编码RNA(miRNA/lncRNA)差异表达谱。2)由于高通量技术结果存在潜在假阳性及假阴性,通过RT-PCR对筛选出的关键差异非编码RNA(miRNA/lncRNA)及mRNA再次验证。再次在上述MELAS(A3243G突变患者)及活检排除肌肉病变的对照者各20例单个肌肉样本中进行RT-PCR实验验证高通量初筛结果,确认关键性ncRNAs(miRNAs、IncRNAs)及相关靶mRNAs在MELAS(A3243G突变患者)肌肉组织中的表达差异。实验结果1.我们首次发现MELAS A3243G突变患者肌肉组织中独特的ncRNAs(miRNAs/lnc RNAs)表达谱,共筛选出具有2倍以上差异的microRNAs(31个上调;41个下调),lncRNAs(57个上调;52个下调)以及mRNAs(167个上调;162个下调);2种线粒体肌病(mtDNA A3243G突变与核基因TK2突变)肌肉组织显示二者共有41个共差异表达的mRNAs。2.我们通过对筛选出的 6 个 miRNAs(miR-6089、miR-27b-3p、miR-214-3p、mi R-150-5p、let-7e-5p 和 miR-145-5p)、5个IncRNAs(LINC01405、SNHG12、R P11-403P17.4、CTC-260E6.6 和 RP11-357D18.1)和 6 个 mRNAs(PDK4、CDKN1A、ATP2A2、SOD3、DDIT4和EEF1A1)进行RT-PCR验证,结果显示同高通量测序结果比较二者基本一致。第二部分线粒体肌病肌肉差异RNA分子互作网络构建及关键ncRNA(miRNA/lncRNA)筛选研究目的构建mRNA及非编码RNA(miRNA/lncRNA)相互间调控网络,并分析其参与的信号通路建立调节机制假说。研究方法对差异ncRNA(miRNA/lncRNA)进行生物信息学(聚类及GO和Pathway分析)、表达相关性等分析确定参与不同突变类型线粒体肌病患者发生发展的关键非编码R NA(miRNA/lncRNA)相互作用分子。共表达互作网络构建包括miRNA-mRNA、miRNA-lncRNA 及 IncRNA-mRNA 两两互作网络以及 miRNA-mRNA-lncRNA 和 mRNA-miRNA-lncRNA三分子互作网络。应用Cytoscape软件计算网络及各节点的拓扑特性。通过miRecords预测差异miRNA的靶基因,对差异靶基因进行GO功能注释、KEGG通路分析。实验结果1.我们首次通过生物信息学靶基因预测构建出miRNA-mmRNA-lncRNA和mRNA-mi RNA-lncRNA共表达互作网络,筛选出调控网络中关键性ncRNA分子。2.GO分析发现差异miRNA/lncRNAs靶基因集合及总体差异mmRNAs集合共同的生物过程富集分析集中于细胞外基质分子、肌肉收缩和蛋白结合分子。除此之外,总的差异表达mRNAs的显著GO条目富集结果集中于小分子代谢、信号转导和转录分子等过程;miRNA靶mRNA网络代表的显著GO分类条目为信号转导分子、细胞凋亡相关分子等;lncRNA靶mRNA对应的相关联的GO分析富集条目为小分子代谢过程和肌肉收缩相关分子。KEGG通路分析表明这些失调的mRNA主要参与小分子代谢过程、细胞外基质生成、肌肉收缩、蛋白结合、信号转导、转录等信号途径和生物功能。综上所述,MELAS患者mtDNA A3243G突变造成的下游差异表达的ncRNA谱可能通过免疫应答、小分子代谢、信号转导、凋亡和发育过程等多种信号途径参与线粒体肌病的发生发展。第三部分线粒体肌病ncRNA(miRNA/lncRNA)生物学标志物的潜在临床应用价值研究目的本课题进一步分析关键性ncRNAs在肌肉组织和血清对MELAS患者的诊断效能,寻找线粒体肌病潜在的ncRNA诊断标志物。研究对象和方法1.选取54例MELAS(A3243G突变患者)与49例正常对照组独立肌肉样本再次验证A3243G突变MELAS患者高通量结果;2.选取34例MELAS(A3243G突变患者)与34例对照独立血清样本验证A3243G突变 MELAS 患者 miR-27b-3p 表达;3.选取22例MELAS(非A3243G突变患者)与34例对照独立肌肉及血清样本验证非 A3243G 突变 MELAS 患者 miR-27b-3p 表达;3.软件分析肌肉组织ncRNAs及血清miR-27b-3p对于MELAS患者诊断的敏感度、特异度;对血清miRNA-27b-3p表达水平与各类临床参数如年龄、性别、BMI、NMDAS、疾病持续时间、血乳酸和肌肉A3243G突变率进行相关性分析。实验结果1.我们再次证实 4 个 ncRNAs(miR-6089、miR-27b-3p、miR-214-3p 及 LINC01405)肌肉表达显著下调,而 7 个ncRNAs(miR-150-5p、let-7e-5p、miR-145-5p、SNHG12、RP11-403P17.4、CTC-260E6.6 及 RP11-357D18.11)在 MELAS 肌肉组织中显著上调;ROC曲线分析表明在所选择的肌肉组织关键性ncRNA中miR-27b-3p对于MELAS患者诊断具有最高的灵敏度和特异性。2.关键性miR-27b-3p在MELAS患者(A3243G和非A3243G突变患者)肌肉和血清中表达均降低。3.ROC曲线分析表明,相比乳酸血清miR-27b-3p对于MELAS具有更好的诊断价值。相关性分析显示血清miR-27b-3p和乳酸盐及NMDAS之间存在显著负相关。结论我们首次发现MELAS A3243G突变患者肌肉组织中独特的ncRNAs(miRNAs/lncRNAs)表达谱;首次构建出 miRNA-mRNA-lncRNA 和 mRNA-miRNA-lncRNA共表达互作网络,筛选出调控网络中关键性ncRNA分子血清miR-27b-3p比乳酸对于MELAS具有更好的诊断价值,并通过功能富集分析明确了非编码RNA分子参与线粒体肌病可能主要通过信号转导、氧化应激、凋亡等途径,为进一步探究相应ncRNA分子的功能提供理论依据。
[Abstract]:Mitochondrial DNA (mtDNA) DNA and / or nuclear mitochondrial function related protein encoding gene deletions or point mutations caused by encoding mitochondrial oxidative metabolism enzymes essential carrier or impairment of glycogen and fatty acids into mitochondria can not fully utilized, resulting in energy metabolism and has complex clinical symptoms called mitochondrial diseases (mitochondrial disease). Mitochondrial diseases of skeletal muscle involvement called mitochondrial myopathy (mitochondrial myopathy). The clinical type of mitochondrial myopathy involving complex, including mitochondrial myopathy with lactic acidosis and stroke like episodes (MELAS) syndrome and other clinical syndromes, different clinical types tend to have different mutations but also on mitochondria and diseases overlap. And senescence, programmed cell death, the relationship between oxygen free radicals and cellular signal transduction in the process of a Has been the research hotspot in mitochondrial medicine. Gene sequencing progress makes us aware of the human genome, there are tens of thousands of kinds of does not have the function of encoding protein but with gene regulation and apparent non genetically modified RNA encoding function (ncRNA), such as small non encoding RNA (miRNA), RNA (long chain non encoding long non-coding RNA, lncRNA). In recent years, ncRNAs has been proved by several different mechanisms influencing gene expression level, but the vast majority of non RNA encoding function is unknown. More complicated is that all kinds of RNA molecules in the presence of a complex regulatory network between. Thanks to the rapid development of nucleic acid sequencing techniques, more and more the study found that in human serum / plasma in the presence of a large number of stable ncRNA. non RNA encoding can be used as molecular biological markers of disease have all kinds of features, there is a huge potential to become mitochondrial myopathy The diagnosis and treatment of markers of disease. Research on mitochondrial function of ncRNA is still at the start stage, the influence of ncRNA to the mitochondrial gene expression and function is a new field. More and more studies have confirmed that RNA can express non encoding or regulation of mitochondrial genes directly in their mitochondria affects mitochondrial function by affecting the expression of nuclear genes encoding mitochondrial egg white. The current study of mitochondrial function related gene RNA encoding is still mostly limited in non related mitochondrial damage in vitro cell model of non encoding RNA discovery and functional verification. Although many have been found to affect mitochondrial function of miRNAs and lncRNAs, but the regulation of mitochondrial function with ncRNA mechanism to be further research shows that.NcRNA has not been reported with mitochondrial myopathy it is necessary for us to study, ncRNA is involved in the occurrence and development mechanism of mitochondrial myopathy with, or Close contact can reveal ncRNA and mitochondrial function and mitochondrial myopathy, find potential therapeutic targets. We hope that the regulation of ncRNA level, ncRNAs found some key control of mitochondrial function or participate in the pathogenesis of mitochondrial myopathy in mitochondrial myopathy, and could achieve the abnormal mitochondria by affecting ncRNA effective removal effect the key to the development of mitochondrial myopathy occurred in the pathological process of regulation. NcRNA molecular markers have the characteristics of disease diagnosis and treatment can also be used as a marker of mitochondrial myopathy diagnosis and treatment of the clinical service. For this, this topic through screening the differentially expressed ncRNA and healthy control in patients with mitochondrial myopathy muscle tissue and serum (miRNA/lncRNA), RNA between different construction control network, explore the potential pathway and mitochondrial ncRNA damage in mitochondrial myopathy on occurrence and development of the key The mechanisms that regulate the expression of IncRNA and ncRNA, to find the key process in the development of mitochondrial myopathy (miRNA/lncRNA), to provide theoretical basis for the diagnosis and treatment of the disease is ncRNA for muscle mitochondria. The issue is about the non regulation mechanism of mitochondrial disease encoding RNA involved in mitochondrial myopathy and development are as follows: first part of muscle mitochondrial myopathy (MI RNA/IncRNA) RNA encoding pre screening strategy to study the spectrum of high throughput technology combined with subsequent RT-PCR repetitive test of differential expression screening of representative mitochondrial encephalomyopathy MELAS (A3243G mutation) encoding RNA non muscle tissues (miRNA/lncRNA) expression profile differences, provide a basis for interested to follow the ncRNA and complex mechanism of non encoding RNA involved in mitochondrial myopathy. Subjects and methods 1 selected MELAS (A3243G) first Mutation) and biopsy to exclude muscle disease control group 20 cases, using miRNA microarray and high-throughput IncRNA sequencing technology, MELAS screening (A3243G mutation) - encoding RNA compared with control muscle tissue (miRNA/lncRNA) expression difference of.2) due to the high throughput technology the potential false positive and false negative. The RT-PCR of the key differences between the selected non encoding RNA (miRNA/lncRNA) and mRNA in the MELAS. Verify again again (A3243G mutation) were excluded and biopsy of muscle lesions in 20 cases of single muscle samples of RT-PCR experimental verification of high-throughput screening results, confirm the key ncRNAs (miRNAs, IncRNAs) and related the target mRNAs in MELAS (A3243G mutation) differentially expressed in muscle tissue. The experimental results of 1. we first found that MELAS A3243G mutation unique muscle tissues of patients with ncRNAs (miRNAs/lnc RNAs) expression, Co Screening is more than 2 times the difference of microRNAs (31 up-regulated; 41 down), lncRNAs (57 up-regulated and 52 down regulated; (mRNAs) 167 up-regulated; 162 down regulated); 2 (mtDNA A3243G TK2 mutation of mitochondrial and nuclear genes mutation) muscle tissue shows that the two total 41 a total of mRNAs.2. of the difference through our 6 selected by miRNAs (miR-6089, miR-27b-3p, miR-214-3p, MI, R-150-5p, let-7e-5p and miR-145-5p), 5 IncRNAs (LINC01405, SNHG12, R, P11-403P17.4, CTC-260E6.6 and RP11-357D18.1) and 6 mRNAs (PDK4, CDKN1A, ATP2A2, SOD3, DDIT4 and EEF1A1) RT-PCR verification the results show, with high-throughput sequencing of the two are basically the same. The second part of mitochondrial myopathy muscle differences of RNA molecular interaction network construction and key ncRNA (miRNA/lncRNA) screening research objective to construct mRNA and non RNA encoding (miRNA/lncRNA) between control network The network, and the signal pathway involved in the establishment of regulation mechanism hypothesis. Research methods on the difference of ncRNA (miRNA/lncRNA) by Bioinformatics (cluster analysis and GO and Pathway), correlation analysis to determine the expression in different key types of mutation in patients with mitochondrial myopathy of the occurrence and development of non encoding R NA (miRNA/lncRNA) co expression of molecular interactions. The interaction network including miRNA-mRNA, miRNA-lncRNA and IncRNA-mRNA 22 interaction network as well as miRNA-mRNA-lncRNA and mRNA-miRNA-lncRNA three molecular interaction network. Computing the topological properties of the network and the nodes using Cytoscape software by miRecords. The predicted target gene differences in miRNA, GO functional annotation of different target genes, KEGG pathway analysis. The experimental results of 1. for the first time bioinformatics predicted target gene construct the interaction network of co expression of miRNA-mmRNA-lncRNA and mRNA-mi RNA-lncRNA, Selected key ncRNA molecular regulatory network.2.GO analysis showed that the difference of miRNA/lncRNAs target gene set and mmRNAs set the overall difference of biological processes common enrichment analysis focused on extracellular matrix molecules, muscle contraction and protein binding molecules. In addition, mRNAs was a GO entry enrichment were focused on small molecule metabolite expression total difference signal molecular transduction and transcription process; significant GO classification entry miRNA mRNA network on behalf of the target for signal transduction, apoptosis related molecules; analysis of corresponding lncRNA target mRNA associated GO enrichment entry into small molecule metabolism and muscle contraction related molecules in.KEGG pathway analysis showed that the imbalance of mRNA mainly involved in small molecule metabolism, extracellular matrix formation, muscle contraction, protein binding, signal transduction, transcription, signal pathways and biological functions. In conclusion, MELAS in patients with MT The expression of downstream differences caused by mutations in the DNA A3243G ncRNA spectrum by immune response, small molecule metabolism, signal transduction, apoptosis and development process of the occurrence and development of a variety of signaling pathways involved in mitochondrial myopathy. The third part ncRNA of mitochondrial myopathy (miRNA/lncRNA) objective to study the clinical application value of potential biological markers of this topic for further analysis of key ncRNAs the diagnostic efficacy of muscle tissue and serum of MELAS patients, looking for mitochondrial potential biomarker for diagnosis of ncRNA. The research object and methods 1. a total of 54 cases of MELAS (A3243G mutation) and 49 cases of normal control group independent muscle samples tested again A3243G mutation in patients with MELAS high throughput results; 2. (a total of 34 cases of MELAS patients with A3243G mutations) and 34 cases of control independent verification of A3243G mutations in MELAS patients serum samples from 22 cases of miR-27b-3p expression; 3. (non MELAS patients with A3243G mutation) 34 cases of control independent muscle and serum samples to verify the non A3243G mutation in patients with MELAS miR-27b-3p expression; analysis of muscle tissue ncRNAs and serum miR-27b-3p for the diagnosis of patients with MELAS sensitivity, specificity 3. software; expression levels and various clinical parameters such as age and gender on serum miRNA-27b-3p, BMI, NMDAS, disease duration, blood lactic acid and muscle A3243G relationship between the mutation rate were analyzed. The experimental results confirmed that 1. of our 4 ncRNAs (miR-6089, miR-27b-3p, miR-214-3p and LINC01405) muscle expression were significantly reduced, while the 7 ncRNAs (miR-150-5p, let-7e-5p, miR-145-5p, SNHG12, RP11-403P17.4, CTC-260E6.6 and RP11-357D18.11) were significantly up-regulated in MELAS muscle tissue; ROC curve analysis showed that the key of ncRNA the selected muscle tissue miR-27b-3p with miR- key sensitivity and specificity for the diagnosis of MELAS patients with the highest.2. 27b-3p in MELAS patients (A3243G and non A3243G mutation) in serum and muscle decreased the expression of.3.ROC curve analysis showed that, compared with the diagnostic value of serum lactic acid miR-27b-3p is better for MELAS. Correlation analysis showed that there is significant negative correlation between serum miR-27b-3p and lactate and NMDAS. Conclusion we first found that MELAS A3243G mutation ncRNAs unique muscle tissue patients (miRNAs/lncRNAs) expression profile; for the first time to construct miRNA-mRNA-lncRNA and mRNA-miRNA-lncRNA co expression interaction network, selected key ncRNA molecular network in serum miR-27b-3p has better diagnostic value than lactic acid for MELAS, and through the functional enrichment analysis identified non encoding RNA molecules involved in mitochondrial myopathy may be mainly through signal transduction, oxidative stress. Apoptosis, provide a theoretical basis for the further exploration of the corresponding ncRNA molecular function.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R596

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